Mycobacterium avium subspecies paratuberculosis (hereinafter referred to as M. paratuberculosis) causes Johne's disease (paratuberculosis) in dairy cattle and other animals characterized by chronic diarrhea, weight loss, and malnutrition, resulting in estimated losses of $220 million per year in the U.S.A. alone. World-wide, the prevalence of the disease can range from as low as 3-4% of the examined herds in regions with low incidence (such as England), to high levels of 50% of the herds in some areas within the U.S.A. (Wisconsin and Alabama). Cows with Johne's disease excrete Mycobacterium paratuberculosis in their milk. In humans, M. paratuberculosis bacilli have been found in tissues examined from Crohn's disease patients indicating possible zoonotic potential of this pathogen.
Diagnosis of bovine paratuberculosis is difficult, and is typically accomplished by detecting either the causative agent M. paratuberculosis or an immune response to the agent. Many clinical methods for detecting and identifying Mycobacterium species in samples require analysis of the bacterium's physical characteristics, physiological characteristics, or biochemical characteristics. These methods require relatively high concentrations of bacteria in the sample to be detected, may be subjective depending on the clinical technician's experience and expertise, and are time-consuming. Because Mycobacterium species are often difficult to grow in vitro and may take weeks to reach a useful density in culture, these methods can also result in delayed diagnosis and intervention to stop the spread of infection. For example, microbiological culture of Mycobacterium from feces is a widely used diagnostic test; however, this assay requires up to 16 weeks for completion.
Commercially available diagnostic tests exist. For example, serologic tests based on Enzyme-Linked Immunoabsorbent Assay (ELISA) technology are popular commercially available immunoassays. ELISA technology is based upon the use of an enzyme-linked antibody marker to detect the presence of specimen antibody bound to a known antigen that is attached to a solid support. However, the accuracy of existing commercially available ELISA kits for bovine paratuberculosis is relatively poor. A comparison of commercial ELISA kits showed that assays performed comparably overall with diagnostic sensitivity ranging from 27.9% to 44.5% for fecal culture-positive cattle (Collins et al., 2005, Clin. Diagn. Lab. Immunol. 12: 685-692). In a more recent study, the commercially-available ELISAs for M. paratuberculosis were found to have an even lower sensitivity of approximately 13.5% (Sweeney et al., 2006, J. Vet. Diagn. Invest. 18: 2-6). Accordingly, there is a need in the art for paratuberculosis diagnostic tests based on immune response detection.